Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Third Military Medical Univ., Chongqing 400038, China.
Am J Physiol Heart Circ Physiol. 2010 Jun;298(6):H1919-31. doi: 10.1152/ajpheart.01039.2009. Epub 2010 Mar 12.
Hypoxia-inducible factor (HIF)-1alpha is a key regulator of anaerobic energy metabolism. We asked the following question: Does the breakdown of microtubular structures influence glycolysis in hypoxic cardiomyocytes by regulating HIF-1alpha? Neonatal rat cardiomyocytes were cultured under hypoxic conditions, while microtubule-stabilizing (paclitaxel) and -depolymerizing (colchicine) agents were used to change microtubular structure. Models of high microtubule-associated protein 4 (MAP4) expression and RNA interference of microtubulin expression were established. Microtubular structural changes and intracellular HIF-1alpha protein distribution were observed with laser confocal scanning microscopy. Content of key glycolytic enzymes, viability, and energy content of cardiomyocytes were determined by colorimetry and high-performance liquid chromatography. HIF-1alpha protein content and mRNA expression were determined by Western blotting and real-time PCR, respectively. Low doses of microtubule-stabilizing agent (10 mumol/l paclitaxel) and enhanced expression of MAP4 stabilized the reticular microtubular structures in hypoxic cardiomyocytes, increased the content of key glycolytic enzymes, ameliorated energy supply and enhanced cell viability, and upregulated HIF-1alpha protein expression and endonuclear aggregation. In contrast, the microtubule-depolymerizing agent (10 mumol/l colchicine) or reduced microtubulin expression had adverse affects on the same parameters, in particular, HIF-1alpha protein content and endonuclear aggregation. We conclude that microtubular structural changes influence glycolysis in the early stages of hypoxia in cardiomyocytes by regulating HIF-1alpha content. Stabilizing microtubular structures increases endonuclear and total HIF-1alpha expression, content of key glycolytic enzymes, and energy supply. These findings provide potential therapeutic targets for ameliorating cell energy metabolism during early myocardial hypoxia.
缺氧诱导因子 1α（HIF-1α）是无氧能量代谢的关键调节因子。我们提出了以下问题：微管结构的破坏是否通过调节 HIF-1α影响缺氧心肌细胞的糖酵解？将新生大鼠心肌细胞在缺氧条件下培养，同时使用微管稳定（紫杉醇）和微管解聚（秋水仙碱）剂来改变微管结构。建立高微管相关蛋白 4（MAP4）表达模型和微管表达 RNA 干扰模型。用激光共聚焦扫描显微镜观察微管结构变化和细胞内 HIF-1α 蛋白分布。通过比色法和高效液相色谱法测定心肌细胞关键糖酵解酶含量、活力和能量含量。通过 Western blot 和实时 PCR 分别测定 HIF-1α 蛋白含量和 mRNA 表达。低剂量的微管稳定剂（10 mumol/l 紫杉醇）和 MAP4 的增强表达稳定了缺氧心肌细胞的网状微管结构，增加了关键糖酵解酶的含量，改善了能量供应，增强了细胞活力，并上调了 HIF-1α 蛋白表达和核内聚集。相比之下，微管解聚剂（10 mumol/l 秋水仙碱）或微管蛋白表达减少对相同参数产生了不利影响，特别是 HIF-1α 蛋白含量和核内聚集。我们得出结论，微管结构的变化通过调节 HIF-1α 含量影响心肌细胞缺氧早期的糖酵解。稳定微管结构增加核内和总 HIF-1α 表达、关键糖酵解酶含量和能量供应。这些发现为改善早期心肌缺氧时细胞能量代谢提供了潜在的治疗靶点。
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